Linker element and method of using same to construct sequencing library

ABSTRACT

Provided is a linker element and a method of using the linker element to construct a sequencing library, wherein the linker element consists of a linker A and a linker B, the linker A is obtained through the complementary pairing of a long nucleic acid strand and a short nucleic acid strand, the 5′ end of the long strand has a phosphoric acid modification, and the 3′ end of the short strand has an enclosed modification, with enzyme sites in the short strand; and the linker B is a nucleic acid single strand, and the 3′ end thereof can be in a complementary pairing with the 5′ end of the long strand of the linker A. Using the linker element of the present invention for constructing a sequencing library ensures the linking directionality of the linkers while solving the problems of fragment interlinking, linker self-linking and low linking efficiency, and reducing the purification reaction between steps, shortening the linking time and reducing costs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application under 35 U.S.C. § 371of International Application No. PCT/CN2014/088592, filed on Oct. 14,2014, the contents of which are hereby incorporated herein by referencein their entirety.

TECHNICAL FIELD

The present invention relates to the field of biotechnology and, inparticular, to a linker element, a method of constructing a sequencinglibrary using the linker element, the constructed sequencing library andapplication thereof.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 774972000100SEQLIST.txt,date recorded: Apr. 13, 2017, size: 3 KB).

BACKGROUND ART

High-throughput sequencing has become one of the foundations for modernmolecular biology, biotechnology, medicine and other fields. In recentyears, studies on rapid, accurate and economic methods for determininggene expression level and nucleotide sequence have achieved continuousinnovation; the second generation of high-throughput sequencingtechnology with sequencing by synthesis as the basic principle hasbecome mature. The major sequencing companies have focused on thedevelopment of new sequencing products, shortening the process ofsequencing and cost reduction. The currently existing sequencingproducts based on the second-generation sequencing technology includewhole genome resequencing, whole transcriptome sequencing, andsmall-molecule RNA sequencing and the like. In particular, theapplication derived from the second-generation sequencing combined withmicroarray technology—target sequence capture sequencing technology canuse a large number of oligonucleotide probes to complementarily bind tospecific regions in the genome to capture and enrich gene fragments fromthe specific regions for sequencing; and for the detection, diagnosisand research of disease genes.

Complete Genomics (CG) Corporation currently has an independentlydeveloped second-generation sequencing technology suitable for humanwhole genome sequencing. The process for its library constructionincludes: genomic DNA disruption, the first linker ligation,double-strand cyclization and digestion, the second linker ligation,single-strand separation and cyclization, wherein the two linkerligations are very important in the process for library construction.The linker is a specially designed DNA sequence and when fixed to bothends of the DNA fragment by ligation or the like, can be identifiedduring sequencing and used as a starting site for sequencing to enablethe instrument to read the subsequent sequence information. In order toensure that the read sequence information is easy to analyze, it isnecessary to add two different linkers at both ends (5′ and 3′ ends) ofa DNA fragment; in order to achieve this particular directionalligation, while avoiding the interconnection between the linkers, stickyend can be used to link the linkers; however, this requires fragmentswith sticky ends, which make it difficult to avoid the interconnectionbetween fragments. The Complete Genomics Corporation uses multiple stepsto add linkers to both ends respectively for construction of asequencing library. In order to obtain a fragment with linkers at bothends, it is necessary to go through the following five steps: ligating alinker to one end of a DNA fragment; performing denaturation, annealingand extension; then ligating a linker to the other end of the DNAfragment; filling the vacancy; and performing a polymerase chainreaction. However, the multiple extension reactions therein requireexpensive reagents and multiple purification steps are required betweenthe steps, thus resulting in high overall costs and lack of efficiency.

In order to solve the problems that too many steps are required forlinker ligation in the construction of the sequencing platform libraryof Complete Genomics, the time for constructing the whole library is toolong and the cost is too high, the present invention is proposed.

SUMMARY

In view of the above disadvantages of the prior art, it is an object ofthe present invention to provide a linker element, a method ofconstructing a sequencing library using the linker element, theconstructed sequencing library and application thereof. The method forconstructing the sequencing library of the present invention avoids theconventional linker ligation method that adds linkers to both endsrespectively in multi-steps. By means of a linker with a unique sequenceconfiguration, and a novel linker ligation method consisting of linkerligation plus single strand replacement, the method for constructing thesequencing library of the present invention ensures the directionalityof the linker ligation while solving the problems of fragmentinterconnection, linker self-connection and low ligation efficiency, andsuccessfully reduces the whole linker ligation process to four new stepsand reduces the purification reactions between steps, which greatlyreduce the time required for ligating the linkers, and significantlyreduce costs. In addition, the method of constructing a sequencinglibrary also introduces the nucleic acid probe capture technology torealize the sequencing of the target genomic region, and succeeds increating a target region capture sequencing product based on a singlestrand circular sequencing platform.

In a first aspect, the present invention provides a linker elementconsisting of a linker A and a linker B, wherein the linker A isgenerated from the complementary pairing of a long strand of nucleicacid and a short strand of nucleic acid, wherein the long strand has aphosphate modification at the 5′ end and the short strand has a blockingmodification at the 3′ end, and has an enzyme active site in the shortstrand; and the linker B is a single-stranded nucleic acid, and the 3′end thereof can be complementary to the 5′ end of the long strand of thelinker A but the rest part cannot be complementary to the linker A.

Preferably, in the linker A, the long strand has a length of 40-48 bpand the short strand has a length of 9-14 bp.

Preferably, in the linker B, the length complementary to the long strandof the linker A is 6-12 bp, and the length not complementary to the longstrand of the linker A is 9-15 bp.

Preferably, the blocking modification is a dideoxy blockingmodification.

Preferably, the enzyme active site in the short strand is U or dU, andthe corresponding enzyme is User enzyme.

Preferably, the linker B has a tag sequence; due to the presence of thetag sequence, in the subsequent steps, different samples with differenttags can be mixed and placed in the same reaction system for reaction,further saving operating steps and cost.

In one preferred embodiment, the sequence of the long strand of thelinker A is: /Phos/GTCTCCAGTCTCAACTGCCTGAAGCCCGATCGAGCTTGTCT (i.e., SEQID NO: 1), the sequence of the short strand of the linker A isGACUGGAGAC/ddC/(i.e., SEQ NO: 2), the sequence of the linker B isTCCTAAGACCGCACTGGAGAC (i.e., SEQ ID NO: 3), wherein the group inside“//” represents terminal modified group, “Phos” representsphosphorylation and “dd” represents dideoxy.

In a second aspect, the present invention provides a linker ligationmethod, comprising ligating a linker element as described in the firstaspect to both ends of a DNA fragment to be tested.

Preferably, the linker ligation method comprises the following steps:

(1) the linker A is added to both ends of the DNA fragment to be testedby a ligation reaction;

(2) the DNA fragment ligated with the linker A is treated with acorresponding enzyme according to the enzyme active site in the shortstrand;

(3) the linker B is added to both ends of the DNA fragment ligated withthe linker A and treated in the step (2) by a ligation reaction.

Preferably, the steps of dephosphorylating and blunt-end repairing theDNA fragment to be tested are further comprised before ligating thelinker element; in the step (2), it is further preferred that the stepof phosphorylating the unlinked 5′ end of the DNA fragment is furtherincluded, and it is further preferred that the phosphorylation treatmentis carried out using a polynucleotide kinase.

In a third aspect, the present invention provides a method forconstructing a sequencing library, which uses the linker element asdescribed in the first aspect or the linker ligation method as describedin the second aspect to perform linker ligation.

In a preferred embodiment, the method for constructing a sequencinglibrary comprises the steps of:

1) fragmenting the DNA to be tested;

2) dephosphorylating and blunt-end repairing the DNA fragments obtainedin step 1);

3) linker ligations:

linker A ligation: the linker A is added to both ends of the DNAfragments obtained in Step 2) by a ligation reaction;

enzyme treatment and phosphorylation: depending on the enzyme activesite in the short strand of the A linker, the DNA fragments ligated withthe linker A are treated with the corresponding enzyme, and the unlinked5′ ends of the fragments are phosphorylated; linker B ligation: througha ligation reaction, the linker B is added to both ends of the DNAfragments ligated with the linker A;

4) amplification of DNA fragments: polymerase chain reaction is carriedout by using the DNA fragments obtained in step 3) as a template andusing single-stranded nucleic acids C and D, which are complementary tothe long strand of the linker A and the nucleic acid strand of thelinker B, as primers;

5) hybridization capture: the product obtained in step 4) is captured byhybridizing with an oligonucleotide probe and in the enrichment step ofthe hybridization product, a separation marker is introduced at the 5′end of one strand of the double-stranded nucleic acid and a phosphategroup modification is introduced at the 5′ end of the other strand;

6) separation and cyclization of single-stranded nucleic acids: theproduct obtained in step 5) is separated by utilizing the separationmarker to obtain another nucleic acid single strand without theseparation marker; and a single strand circular nucleic acid product isobtained by cyclizing the obtained nucleic acid single strand, that isthe sequencing library.

Regarding the above method for constructing a sequencing library:

In step 1), preferably, the DNA to be tested is genomic DNA.

Preferably, the fragmentation is a random disruption of the DNA to betested using a physical or chemical method.

Preferably, the fragmentation of the DNA to be tested is performed byusing physical ultrasound or enzymatic reaction.

Preferably, the length of the DNA fragment is 150-250 bp.

In step 2), preferably, the dephosphorylation is carried out by usingalkaline phosphatase, preferably shrimp alkaline phosphatase.

Preferably, the blunt-end repair is performed by using T4 DNApolymerase.

In a preferred embodiment, the sequence of the long strand of the linkerA in step 3) is /Phos/GTCTCCAGTCTCAACTGCCTGAAGCCCGATCGAGCTTGTCT (i.e.,SEQ ID NO: 1), the sequence of the short strand of the linker A isGACUGGAGAC/ddC/ (i.e., SEQ ID NO: 2), and the sequence of the linker Bis TCCTAAGACCGCACTGGAGAC (i.e., SEQ ID NO: 3), wherein the group in “//”is a terminal modified group, “Phos” represents phosphorylation, and“dd” represents dideoxy. In a further preferred embodiment, in step (4),the sequence of the single-stranded nucleic acid C is/Phos/AGACAAGCTCGATCGGGCTTC (i.e., SEQ ID NO: 4), the sequence of thesingle-stranded nucleic acid D is TCCTAAGACCGCACTGGAGAC (i.e., SEQ IDNO: 5), wherein the group in “//” is a terminal modified group, “Phos”represents phosphorylation.

In step 5), preferably, the oligonucleotide probe is a library ofoligonucleotide probes; the hybridization capture step using anoligonucleotide probe allows the sequencing library of the presentinvention to achieve full exome sequencing. Further, by changing theoligonucleotide probe used, other different sequencing requirements canbe met.

Preferably, the separation marker is a biotin modification.

In step 6), preferably, the step of removing the remaining uncyclizedsingle strands by treatment with an exonuclease or the like is alsoincluded after cyclization of the single-stranded nucleic acid.

The single-stranded circular nucleic acid product obtained by theabove-mentioned construction method can be used directly in thesubsequent sequencing step in which the single-stranded circular nucleicacids are subjected to rolling replication to form nucleic acidnanospheres (DNB) for reading nucleic acid sequence information.

In a fourth aspect, the present invention provides a sequencing library,which is prepared by the construction method as described in the thirdaspect.

In a fifth aspect, the present invention provides the use of thesequencing library as described in the fourth aspect for genomicsequencing, preferably for sequencing of a target genomic region;preferably, the sequencing is performed by using a single-strandedcircular library sequencing platform; more preferably, the sequencing isperformed by using sequencing platform of Complete Genomics.

In a sixth aspect, the present invention provides a nucleic acidsequencing method comprising the step of sequencing the sequencinglibrary as described in the fourth aspect; preferably, the sequencing isperformed by using a single-stranded circular library sequencingplatform; further preferably, the sequencing is performed by usingsequencing platform of Complete Genomics; preferably, the method furthercomprises the step of assembling and/or splicing the sequencing results.

In a seventh aspect, the present invention provides a sequencing libraryconstruction kit comprising a linker element as described in the firstaspect.

Preferably, the kit further comprises a dephosphorylase, preferably analkaline phosphatase, more preferably a shrimp alkaline phosphatase; aDNA polymerase, preferably a T4 DNA polymerase; a User enzyme; and aphosphorylase, preferably a polynucleotide kinase.

Beneficial Effect

After the treatment in step 2), wherein the target nucleic acid fragmentundergoes the terminal-blocking treatment of dephosphorylation, thefragmented DNA to be tested becomes a blunt-end fragment with both endsblocked, so that the interaction between the fragments is completelyprevented, and thus the utilization of DNA fragments prior to ligationis extremely highly guaranteed.

The present invention introduces a phosphate group at the 5′ end of thelong strand of the linker A and a blocking modification at the 3′ end ofthe short strand of the linker A. The blocked end cannot be ligated withthe target nucleic acid fragment due to the presence of the blockingmodification. Due to the special construction of the long and shortstrands themselves, there is no interconnection between the linkers,thus ensuring that the 5′ end of the long strand of the linker can beaccurately attached to the 3′ end of the target fragment. This design isvery effective in preventing the occurrence of linker interconnection,ensuring the efficiency of the ligation reaction.

In the target fragment phosphorylation step as designed to performfollowing the ligation of linker A, one end of the target fragment whichis not linked to linker is phosphorylated. The short strand of thelinker A is shortened and falls off during the enzymatic treatment afterthe ligation of the linker A, so that the linker B can be partiallypaired to the long strand of the linker A. The above all make itpossible to orientate the linker B, and ensure the directionality of thelinker ligation. In the conventional linker ligation step of CompleteGenomics, after the ligation of linker A, denaturation, annealing andextension are performed to avoid ligating the same linker to both ends(as shown in FIG. 2, No. 1). Although this method also guarantees thedirectionality of the linker ligation, it needs to use high-fidelityhot-start enzyme, which incurs high cost, and long reaction time.However, the treatment enzymes (such as the User enzyme) used in theligation method of the present invention are relatively inexpensive andrequire mild enzymatic reaction conditions, and the requirement to thereaction system is low, and the purification treatment step can beomitted before the enzyme treatment. Overall, the present inventionreduces costs and processing time.

In the ligation of B linker, the characteristics of the short and longstrands of linker A are also cleverly utilized. Since after the enzymetreatment, there are less and unstably bonded complementary pairingbases between the short stand and the long strand, the short strand willbe separated from the long strand at relatively milder temperatures. Thesingle strand of the linker B having a longer complementary base pairingsequence and a more dominant binding ability is simply complementary tothe long strand of the linker A so that make it precisely linked to thevacant end of the target fragment. The other parts of the linker B thatis not complementary ensure the differences between linker A and B. Bysubsequent polymerase chain reaction, the target fragment with differentterminal sequences (i.e., the long strand of the linker A at one end,and the linker B at the other end) is ultimately formed. This uniquedesign, in combination with polymerase chain reaction, solves theproblems of cost-effective introduction of different linkers at bothends of the fragment in the blunt-end ligation. It also avoids theoccurrence of fragment/linker interactions resulting from the ligationat cohesive end of the fragment produced by a step of introducingadenylate at terminal.

Compared with the traditional ligation method with linker B (shown inFIG. 2, No. 2) of Complete Genomics, this unique partially base-pairedsingle strand linker design allows the linker ligation and vacancyfill-up to be replaced by a single step, greatly reducing the processand saving cost.

Based on the traditional sequencing library construction scheme ofComplete Genomics, the present invention proposes a sequencing libraryconstruction scheme based on novel linker structure and linker ligationmethod, and introduces a probe hybridization capture step, so as todevelop a novel target region capture library sequencing product basedon the single-stranded circular library sequencing platform, realizingsmall region capture library sequencing based on the single-strandedcircular library sequencing platform from scratch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a sequencing library construction scheme of thepresent invention; 1 represents a disrupted DNA fragment, 2 represents adephosphorylated, terminal-repaired fragment (each terminal is ahydroxyl group), 3 represents the long and short strands of the linkerA, 4 represents the single strand of the linker B, and 5 represents alibrary construction final product of nucleic acid single strand loop.

FIG. 2 illustrates the conventional linker ligation method of CompleteGenomics; 1 represents the treatment step between the ligations oflinker A′ and B′; 2 represents three steps related to linker B′ligation.

DETAILED DESCRIPTION

In order to facilitate understanding of the present invention, thepresent invention is exemplified as follows. It should be apparent tothose skilled in the art that the described examples are merely toassist in understanding the present invention and should not beconstrued as limiting the invention thereto in any way.

EXAMPLE 1 Construction of a Sequencing Library of the Present Invention

1. Disruption of genomic DNA: There are many ways for genomic DNAdisruption, such as physical ultrasound or enzymatic reaction, either ofwhich has very mature schemes on the market. The present example employsa physical ultrasonic disruption method.

A Teflon line and 1 μg of genomic DNA were added into a 96-well PCRplate in turn, and then TE buffer solution or enzyme-free water wasadded to make up 80 μl. The plate was sealed and placed on an E220ultrasonic disruption instrument. The conditions for disruption were setas follows:

Filling coefficient 20% Severe degree  5 Pulse coefficient 200Disruption time 60 s, 5 times

2. Recovery of disrupted fragments: magnetic beads purification methodor gel recovery method can be used. Magnetic bead purification methodwas used in this example.

80 μl of Ampure XP magnetic beads were added into the disrupted DNA, andthen mixed well and placed for 7-15 min; then the mixture was put into amagnetic frame, and the supernatant was collected and added 40 μl ofAmpure XP beads, and then mixed well and placed for 7-15 min; then themixture was put into the magnetic frame, then the supernatant wasremoved, and the magnetic beads were washed twice with 75% ethanol;after drying, the beads was added 50 μl of TE buffer solution orenzyme-free water, and then mixed well and placed for 7-15 min todissolve the recovered product.

3. Dephosphorylation reaction: a system was prepared according thefollowing table using the recovered products of the previous step:

10x NEB Buffer 2 2.4 μl Shrimp alkaline 2.4 μl phosphatase (1 U/ul)Total 4.8 μl

4.8 μl of reaction system was added to the recovered product of theprevious step, mixed, and a reaction was carried out under theconditions listed in the following table. The reaction product was useddirectly for the next step.

37° C. 45 min 65° C. 10 min

4. End repairing of fragments: a system was prepared according to thefollowing table:

Enzyme-free water  4.9 μl 10x NEB Buffer 2 0.72 μl 0.1M adenosine 0.32μl triphosphate 25 mM 0.32 μl deoxyribonucleoside triphosphate Bovineserum albumin 0.16 μl T4 deoxyribonucleic acid  0.8 μl polymerase (3U/ul) Total  7.2 μl

After mixing, the system was added to the product of the previous step,mixed well and incubated at 12° C. for 20 min Purification was performedwith 90 μl of Ampure XP magnetic beads and 18 μl of TE buffer solutionwas used to dissolve the recovered product. (There are many ways topurify the reaction product, such as magnetic bead method, columnpurification method, gel recovery method, etc. All the methods can beused interchangeably. The present example was purified by a magneticbead method unless otherwise specified.)

5. Linker A ligation: The linker-related sequences used in this schemewere as follows (in the sequence, from left to right is the 5′ end tothe 3′ end, the group inside “II” is terminal-modified group, “phos”represents phosphorylation, “dd” represents dideoxy, and “bio”represents biotin):

Long strand of the linker A:

/Phos/GTCTCCAGTCTCAACTGCCTGAAGCCCGATCGAGCTTGTCT  (i.e., SEQ ID NO: 1);

Short strand of the linker A:

GACUGGAGAC/ddC/ (i.e., SEQ ID NO: 2);

The ligation buffer 1 used in this scheme was formulated as follows:

Tris (hydroxymethyl) 150 mM aminomethane-hydrochloric acid (pH 7.8)Polyethylene glycol 8000 15% Magnesium chloride  30 mM Ribonucleosidetriphosphate  3 mM

A system was prepared as follows:

Enzyme-free water 11 μl Linker A (100 uM) 1 μl Ligation buffer 1 13 μlT4 DNA ligase (fast) (600 U/[mu] 1 μl 1) (enzymatics, L6030-HC-L) Total21.5 μl

The above system and the previous product were mixed and reactedaccording to the following table:

25° C. 20 min 65° C. 10 min

6. Phosphorylation and uracil removal: a system was prepared accordingto the following table:

User enzyme (1000 U/ml) 0.5 μl Polynucleotide kinase 0.5 μl (10 U/uL)Total   1 μl

The above system was added to the product of step 5, mixed well andplaced at 37° C. for 15 min.

Purification was performed by using 60 μl of Ampure XP magnetic beads,and 62.5 μl of enzyme-free water or TE buffer solution was used forrecovery.

7. Linker B ligation:

The sequence of linker B was as follows:

TCCTAAGACCGCACTGGAGAC (i.e., SEQ ID NO: 3)

A system was prepared as follows:

Ligation buffer 1 33 μl T4 DNA ligase (fast) (600 U/ 1 μl [mu] 1)(enzymatics, L6030-HC-L) Linker B (100 uM) 1.67 μl Total 37 μl

The above system was added to the recovered product in step 6 and mixedwell and reacted for 20 min at 20° C.

Purification was performed by using 120 μl of Ampure XP magnetic beads,and 45 μl of TE buffer solution was used to dissolve the recoveredproduct.

8. Polymerase chain reaction:

The sequence of primer C was as follows:

/phos/AGACAAGCTCGATCGGGCTTC (i.e., SEQ ID NO: 4)

The sequence of primer D was as follows:

TCCTAAGACCGCACTGGAGAC (i.e., SEQ ID NO: 5)

A system was prepared as follows:

enzyme-free water 45 μl 10x PfuTurbo Cx buffer 100 μl (Agilent,01.Agilent.600414) PfuTurbo Cx hot-start nucleic 2 μl acid polymerase(2.5 U/ul) (Agilent, 01.Agilent.600414) 20 uM primer C 4.0 μl 20 uMprimer D 4.0 μl Total volumn 155.0 μl

The recovered product in the previous step was added to the abovesystem, mixed well, and then reacted according to the conditions listedin the following table:

95° C.  3 min 95° C. 30 s 56° C. 30 s 72° C. 90 s Steps 2-4 wererepeated for 7 times 68° C.  7 min

After completion of the reaction, 240 μl of Ampure XP magnetic beadswere used for purification, and 25 μl of enzyme-free water was used todissolve the recovered product.

9. Hybridization capture: 500 ng-1 μg of reaction product of theprevious step was concentrated and evaporated, and then added to thefollowing system 1 to dissolve:

Blocking Sequence 1:  GAAGCCCGATCGAGCTTGTCT (i.e., SEQ ID NO: 6)Blocking sequence 2:  GTCTCCAGTC (i.e., SEQ ID NO: 7)Blocking Sequence 3:  GTCTCCAGTGCGGTCTTAGGA (i.e., SEQ ID NO: 8)

Enzyme-free water 3.4 μl SureSelect Block # 1 2.5 μl (Agilent)SureSelect Block # 2 2.5 μl (Agilent) Blocking sequence 1 0.3 μlBlocking sequence 2 0.3 μl Blocking sequence 3 0.3 μl Total volume 9.3μl

The mixed reaction system 1 was allowed to react at 95° C. for 5 minutesand kept at 65° C.

System 2 was prepared as follows:

SureSelect Hyb # 1 8.3 μl (Agilent) SureSelect Hyb # 2 0.3 μl (Agilent)SureSelect Hyb # 3 3.3 μl (Agilent) SureSelect Hyb # 4 4.3 μl (Agilent)Total volume 16.3 μl 

System 2 was added to System 1 and kept at 65° C.

System 3 was prepared as follows:

Enzyme-free water 1 μl SureSelect RNase Block 1 μl (Agilent) SureSelectOligo Capture 5 μl Library Total volume 7 μl

System 3 was added to the system 1 and 2, and reacted at 65° C. for20-24 h.

After completion of the reaction, streptavidin-coated magnetic beadswere used for binding, and the beads were dissolved in 50 ul ofenzyme-free water after completion of the binding.

The following reaction system was prepared:

The sequence of primer D with biotin-modification was as follows:

/bio/TCCTAAGACCGCACTGGAGAC (i.e., SEQ ID NO: 9)

Enzyme-free water 40 μl 10x PfuTurbo Cx buffer 100.0 μl (Agilen,01.Agilent.600414) PfuTurbo Cx Hot Start 2 μl Nucleic Acid Polymerase(2.5 U/ul) (Agilent, 01.Agilent.600414) 20 uM primer C 4 μl 20 uM primerD 4 μl (biotin-modification) Total volumn 150 μl

The dissolved magnetic beads were added to the reaction system, mixed,and reacted according to the following table:

95° C.  3 min 95° C. 30 s 56° C. 30 s 72° C. 90 s 68° C.  7 min

After completion of the reaction, 240 μl of Ampure XP beads were usedfor purification. 80 μl of TE buffer solution or enzyme-free water wasused for dissolving the recovered product.

10. Separation of the single-stranded nucleic acids: Streptavidin-coatedbeads were used to bind the biotin-containing target fragments obtainedin Step 9. The single-stranded nucleic acids with no magnetic beadsbound were separated by using 78 μl of 0.1 M sodium hydroxide, and theseparated product was neutralized by the addition of an acidic buffer.The total volume of the neutralized product was 112 μl.

11. Cyclization of the single-strand nucleic acids: The followingreaction system 1 was prepared: wherein the nucleic acid single strand Ehas a corresponding complementary sequence for ligating to both ends ofthe single strand. The sequence of single strand E was as follows:

TCGAGCTTGTCTTCCTAAGACCGC (i.e., SEQ ID NO: 10)

Enzyme-free water 43 μl Nucleic acid single strand E 20 μl Total 63 μl

Reaction system 1 was added to the single strand product of step 10 andmixed.

Preparation of reaction system 2:

Enzyme-free water 153.3 μl 10× TA buffer 35 μl (epicenter) 100 mMAdenosine triphosphate 3.5 μl T4 DNA Ligase (fast) 1.2 μl (600 U/ul)(enzymatics, L6030-HC-L) Total 175 μl

The reaction system 2 was added to the reaction system 1, mixed, andincubated at 37° C. for 1.5 h.

12. Treatment by Exonuclease 1 and Exonuclease 3:

Preparation of the following reaction buffer:

Enzyme-free water 1.5 μl 10× TA buffer 3.7 μl (Epicentre) Exonuclease 1(20 U/ul) (NEB 11.1 μl  Company, M0293S) Exonuclease 3 (100 U/ul) 7.4 μl(NEB Company, M0206S) Total 23.7 μl 

23.7 μl of the reaction buffer was added to 350 μl of the reactionproduct from Step 11, mixed well and incubated at 37° C. for 30 min.

15.4 μl of 500 mM ethylenediaminetetraacetic acid was added and mixedwell. 800 μl of Ampure XP magnetic beads were used for purification and40-80 μl of enzyme-free water/TE buffer was used for dissolving.

The concentrations and total amounts of the final products of thepresent example were as follows:

Total concentration amount (ng/μl) (ng) Product 1 0.40 16 Product 2 0.4216.8 Product 3 0.48 19.2

Applicant declares that the present invention describes the detailedprocess equipment and process flow of the present invention by way ofthe above-described embodiments, however, the present invention is notlimited to the detailed process equipment and process flow describedabove, that is to say, it does not imply that the present invention mustrely on the above-described detailed process equipment and process flow.It should be apparent to those skilled in the art that any modificationof the invention, equivalents of the ingredients of the product of thepresent invention, the addition of auxiliary ingredients, selection ofspecific modes, etc., fall within the disclosed scope and protectivescope of the present invention.

The invention claimed is:
 1. A method for constructing a sequencinglibrary which uses a linker element consisting of a linker A and alinker B, wherein the linker A is generated from the complementarypairing of a long strand of nucleic acid and a short strand of nucleicacid, wherein the long strand has a phosphate modification at the 5′ endand the short strand has a blocking modification at the 3′ end, and hasan enzyme active site in the short strand; and the linker B is asingle-stranded nucleic acid, and the 3′ end thereof can becomplementary to the 5′ end of the long strand of the linker A but therest part cannot be complementary to the linker A, wherein the methodcomprises the steps of: (1) fragmenting a DNA to be tested; (2)dephosphorylating and blunt-end repairing the DNA fragments obtained instep 1); (3) linker ligations: linker A ligation: the linker A is addedto both ends of the DNA fragments obtained in Step (2) by a ligationreaction; enzyme treatment and phosphorylation: depending on the enzymeactive site in the short strand of the A linker, the DNA fragmentsligated with the linker A are treated with the corresponding enzyme, andthe unlinked 5′ ends of the fragments are phosphorylated; and linker Bligation: through a ligation reaction, the linker B is added to bothends of the DNA fragments ligated with the linker A; and (4)amplification of DNA fragments: polymerase chain reaction is carried outusing the DNA fragments obtained in Step (3) as a template and usingsingle-stranded nucleic acids C and D, which are complementary to thelong strand of the linker A and the nucleic acid strand of the linker B,as primers, upon which steps the sequencing library is obtained.
 2. Themethod for constructing a sequencing library according to claim 1,further comprising the steps of: (5) hybridization capture: the productobtained in Step (4) is captured by hybridizing with an oligonucleotideprobe and in the enrichment step of the hybridization product, aseparation marker is introduced at the 5′ end of one strand of thedouble-stranded nucleic acid and a phosphate group modification isintroduced at the 5′ end of the other strand; and (6) separation andcyclization of single-stranded nucleic acids: the product obtained inStep (5) is separated by utilizing the separation marker to obtainanother nucleic acid single strand without the separation marker; and asingle strand circular nucleic acid product is obtained by cyclizing theobtained nucleic acid single strand, that is the sequencing library. 3.The method for constructing a sequencing library according to claim 1,wherein in Step (2) the dephosphorylation is carried out by using shrimpalkaline phosphatase.
 4. The method for constructing a sequencinglibrary according to claim 1, wherein in Step (5) the oligonucleotideprobe is a library of oligonucleotide probes.
 5. The method forconstructing a sequencing library according to claim 1, wherein in Step(2) the blunt-end repair is performed by using T4 DNA polymerase.
 6. Themethod for constructing a sequencing library according to claim 1,wherein in Step (5) the separation marker is a biotin modification. 7.The method for constructing a sequencing library according to claim 1,wherein the long strand of linker A has a length of 40-48 bases and theshort strand of linker A has a length of 9-14 bases.